Cellulose is the most abundant biopolymer on earth. It is a linear polymer of glucose molecules primarily formed by vascular plants but also by green algae, bacteria, and even tunicates. Bacterial cellulose is frequently found in biofilms, which are sessile bacterial communities encased in a 3-dimensional matrix of polysaccharides,proteinaceousfibers,andnucleicacids.Biofilmbacteriaarelesssusceptibletoanti-microbial treatmentsandareresponsibleforabout80%ofhospital-derivedinfections,therebyposingasignificantriskto human health. Developing novel therapeutics to treat or prevent biofilm infections requires a detailed mechanisticunderstandingofhowthebiofilmconstituents,inparticularpolysaccharides,aresynthesizedand depositedoutsidethecell.Theproposedresearchseekstoprovidethisinformation. Bacterial cellulose biosynthesis is an ideal model system to study the mechanism and regulation of exo- polysaccharide secretion. Gram-negatives produce and secrete cellulose via a multi-subunit complex consisting of the inner membrane BcsA and BcsB subunits, the periplasmic BcsZ hydrolase, as well as the outer membrane subunit BcsC. Our previous work provided detailed mechanistic insights into how the inner membrane-integratedBcsA-Bcomplexelongatesthecellulosechainandtranslocatesthepolymeracrossthe plasmamembrane.Whilecurrentdataexplainhowcelluloseisextended,wecurrentlyhavenoinformationon howcellulosebiosynthesisinitiates.ThisquestionwillbeaddressedbiochemicallyinAim1abyreconstituting theinitiationreactioninvitrofromcell-freeexpressed'uninitiated'cellulosesynthase. BcsA processively elongates cellulose and pushes the polymer into a transmembrane channel formed by its own membrane-spanning region. Structural snapshots of different cellulose synthase states during cellulose synthesisandmembranetranslocationprovideinsightsintoconformationalchangesduringthisprocess.Yeta precise analysis of energetic requirements for and processivity rates of cellulose translocation is currently missing.Wewilladdressthesequestionsonasinglemoleculelevelusinganopticallytrappedandcatalytically activeBcsA-BcomplexinAim1b. PasttheinnermembraneandinGram-negatives,cellulosemustcrosstheperiplasmandtheoutermembrane before reaching the biofilm matrix. This section of the translocation path is most likely formed by a direct interactionofperiplasmicandoutermembranecomponentswiththeBcsA-Bcomplexattheinnermembrane. In Aim 2 we seek to reconstitute outer membrane transport of cellulose from nanodisc and proteoliposome- reconstituted components for detailed kinetic, biochemical, and interaction studies. This information will support our efforts to determine the structure of an inner and outer membrane-spanning cellulose synthase complex as outlined in Aim 3. We will use X-ray crystallography and/or electron microscopy to determine the structureofindividualperiplasmicandoutermembranecomponentsaswellastheircomplexeswithBcsA-B.